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CD8<+> cytotoxic T lymphocyte for treatment of colorectal neoplasms and preparation method thereof

A colorectal tumor, cytotoxic technology, applied in the biological field, can solve the problems of anti-tumor immune function damage, inability to completely cure tumors, etc., and achieve the effect of improving killing efficiency and preventing recurrence and metastasis.

Inactive Publication Date: 2015-06-24
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

With the standardization of traditional tumor treatment methods such as surgery, chemotherapy, and radiotherapy, the survival period of patients with colorectal malignant tumors has been significantly prolonged. damage

Method used

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  • CD8&lt;+&gt; cytotoxic T lymphocyte for treatment of colorectal neoplasms and preparation method thereof
  • CD8&lt;+&gt; cytotoxic T lymphocyte for treatment of colorectal neoplasms and preparation method thereof
  • CD8&lt;+&gt; cytotoxic T lymphocyte for treatment of colorectal neoplasms and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1, CD8 for the treatment of colorectal tumors + Preparation of cytotoxic T lymphocytes

[0040] 1. CEA 610D Preparation of peptides

[0041] CEA natural epitope CEA 605-613 The sequence of is YLSGANLNL. The present invention replaces asparagine (N) with aspartic acid (D) on the 6-position, and adopts solid-phase synthesis method to synthesize new modified CEA antigen peptide (CEA 610D ), the amino acid sequence is YLSGADLNL (SEQ ID NO: 1). The antigen peptide is a polypeptide molecule with high affinity combination with MHC class I molecules.

[0042] 2. Load CEA 610D Preparation of Polypeptide B Cells

[0043] 1. Isolation and identification of B cells

[0044] B cells isolated from peripheral blood mononuclear cells (PBMC) of patients with HLA-A2 positive tumors were separated by CD20 magnetic beads to obtain high-purity (above 90%) B cells.

[0045] Through flow cytometry technology, the purity of B cells in the above sorted cells was identified by f...

Embodiment 2

[0087] Example 2, Anti-tumor in vivo experiment of CEA antigen-specific CTL cell tumor-bearing mice obtained in Example 1

[0088] BALB / C-nu / nu nude mice were provided by the Experimental Animal Laboratory of Cancer Hospital, Institute of Cancer, Chinese Academy of Medical Sciences. The age of the mice is 3-5 weeks, the body weight is 17-20 g, and they are raised in nude mice indoors under the condition of SPF.

[0089] 1. Preparation of colorectal tumor mouse model

[0090] Orthotopic transplantation method: BALB / C-nu / nu nude mice are anesthetized by intraperitoneal injection of pentobarbital sodium, take a mid-abdominal incision, identify the colorectum, and make a 2mm incision in the colorectal serosa facing the intestinal wall, as deep as For the mucosal layer, 2 grains of 1mm 3 The tumor mass (surgical specimen of human colorectal cancer with complete histology) was fixed on the mucosal layer of the intestinal wall, and then the surgical incision was sutured. The growt...

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Abstract

The invention discloses a preparation method of a CD8<+> cytotoxic T lymphocyte for the treatment of colorectal neoplasms. The method provided by the invention comprises the step of co-culturing a CEA610D polypeptide loaded B cell and a CD8<+> T cell in vitro, so that the CD8<+> T cell is activated, thereby obtaining a CD8<+> cytotoxic T lymphocyte for the treatment of colorectal neoplasms, wherein the amino acid sequence of a CEA610D polypeptide is shown in sequence 1 in a sequence table. According to the invention, by preparing a large amount of CEA antigen specific CD8<+> CTL in vitro, and combining with thymosin alpha 1, the actions of the two are complemented so as to achieve a synergistic interaction effect, thereby greatly improving the efficiency of tumor killing; and through killing residual neoplasm cells which can not be eliminated by conventional treatment, the recurrence and metastasis of neoplasms are effectively prevented.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a CD8 drug for treating colorectal tumors + Cytotoxic T lymphocytes and methods for their preparation. Background technique [0002] Colorectal cancer (including colon cancer, rectal cancer, and anal cancer) is a common malignant tumor of the digestive tract. It is the third most common malignant tumor in men and the second in women in the world. There are about 1.2 million new cases and deaths every year. About 600,000 cases. With the development of society and economy, the change of residents' lifestyle, and the intensification of population aging, the incidence and death of colorectal cancer in my country are on the rise, and the age of onset has been advanced. With the standardization of traditional tumor treatment methods such as surgery, chemotherapy, and radiotherapy, the survival period of patients with colorectal malignant tumors has been significantly prolonged. damage. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N5/0781A61K35/17A61K38/22A61P35/00
Inventor 刘静维卢戌王跃
Owner BEIJING BIOHEALTHCARE BIOTECH
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